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Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

Background— Three major processes, constrictive vessel remodeling, intimal hyperplasia (IH), and retarded re-endothelialization, contribute to restenosis after vascular reconstructions. Clinically used drugs inhibit IH but delay re-endothelialization and also cause constrictive remodeling. Here we have examined halofuginone, an herbal derivative, for its beneficial effects on vessel remodeling and differential inhibition of IH versus re-endothelialization. Methods and Results— Two weeks after perivascular application to balloon-injured rat common carotid arteries, halofuginone versus vehicle (n=6 animals) enlarged luminal area 2.14-fold by increasing vessel size (adaptive remodeling; 123%), reducing IH (74.3%) without inhibiting re-endothelialization. Consistent with its positive effect on vessel expansion, halofuginone reduced collagen type 1 (but not type 3) production in injured arteries as well as that from adventitial fibroblasts in vitro. In support of its differential effects on IH versus re-endothelialization, halofuginone produced greater inhibition of vascular smooth muscle cell versus endothelial cell proliferation at concentrations ≈50 nmol/L. Furthermore, halofuginone at 50 nmol/L effectively blocked Smad3 phosphorylation in smooth muscle cells, which is known to promote smooth muscle cell proliferation, migration, and IH, but halofuginone had no effect on phospho-Smad3 in endothelial cells. Conclusions— Periadventitial delivery of halofuginone dramatically increased lumen patency via adaptive remodeling and selective inhibition of IH without affecting endothelium recovery. Halofuginone is the first reported small molecule that has favorable effects on all 3 major processes involved in restenosis.

Atherosclerotic-associated diseases are the leading cause of death in the United States. Despite recent progress, interventional treatments for atherosclerosis can be complicated by restenosis resulting from neo-intimal hyperplasia. We have previously demonstrated that TGF-β and its downstream signaling protein Smad3 ∶ 1) are up-regulated following vascular injury, 2) together drive smooth muscle cell (SMC) proliferation and migration and 3) enhance the development of intimal hyperplasia. In order to determine a mechanism through which TGF-β/Smad3 promote these effects, Affymetrix gene expression arrays were performed on primary rat SMCs infected with Smad3 and stimulated with TGF-β or infected with GFP alone. More than 200 genes were differentially expressed (>2.0 fold change, p

We have previously shown that in the presence of elevated Smad3, transforming growth factor-β (TGF-β) transforms from an inhibitor to a stimulant of vascular smooth muscle cell (SMC) proliferation and intimal hyperplasia (IH). Here we identify a novel mechanism through which TGF-β/Smad3 also exacerbates IH by inhibiting SMC apoptosis. We found that TGF-β treatment led to inhibition of apoptosis in rat SMCs following viral expression of Smad3. Conditioned media from these cells when applied to naive SMCs recapitulated this effect, suggesting an autocrine pathway through a secreted factor. Gene array of TGF-β/Smad3-treated cells revealed enhanced expression of vascular endothelial growth factor (VEGF), a known inhibitor of endothelial cell apoptosis. We then evaluated whether VEGF is the secreted mediator responsible for TGF-β/Smad3 inhibition of SMC apoptosis. In TGF-β/Smad3-treated cells, VEGF mRNA and protein as well as VEGF secretion were increased. Moreover, recombinant VEGF-A inhibited SMC apoptosis and a VEGF-A-neutralizing antibody reversed the inhibitory effect of conditioned media on SMC apoptosis. Stimulation of SMCs with TGF-β led to the formation of a complex of Smad3 and hypoxia-inducible factor-1α (HIF-1α) that in turn activated the VEGF-A promoter and transcription. In rat carotid arteries following arterial injury, Smad3 and VEGF-A expression were upregulated. Moreover, Smad3 gene transfer further enhanced VEGF expression as well as inhibited SMC apoptosis. Finally, blocking either the VEGF receptor or Smad3 signaling in injured carotid arteries abrogated the inhibitory effect of Smad3 on vascular SMC apoptosis. Taken together, our study reveals that following angioplasty, elevation of both TGF-β and Smad3 leads to SMC secretion of VEGF-A that functions as an autocrine inhibitor of SMC apoptosis. This novel pathway provides further insights into the role of TGF-β in the development of IH.